Gene regulation plays a central role in all cellular functions, and alterations in this process have been associated with a number of developmental defects and disease states in humans. Initiation of gene expression requires changes to the chromatin environment of genes and the formation of the pre-initiation complex (PIC) at the promoter. The PIC is formed by the ordered recruitment of multiple general transcription factors (GTFs). Advances over the last five years indicate that chromatin is not simply an inert barrier to transcription, but may also play an important function in the recruitment of specific transcription factors to sites within the genome. Further, the distinction between the functions of GTFs and the chromatin remodeling machinery has been blurred by the discovery that certain GTFs have intrinsic chromatin modifying activities, or can recruit them to promoters. The overall goal of this proposal is to understand how chromatin remodeling complexes and Principal Investigator C components are recruited to repressed promoters, and how coordination of the chromatin remodeling and transcription steps occurs. We are using the DNA damage-responsive ribonucleotide reductase 3 gene (RNR3) of budding yeast to explore the functions of the TFIID complex, which is composed of the TATA-binding protein and 14 associated factors called TAFs. Our efforts over the last funding period revealed that TFIID is required for the recruitment of the SWI/SNF nucleosome remodeling complex and the remodeling of nucleosomes at the promoter of RNR3. The objectives of this proposal are to identify the features of RNR3 that confer its dependence upon TAFIIS and the general transcription machinery for nucleosome remodeling. The aims of this proposal will address the role of transcription and the core promoter, identify the components of the pre-initiation complex (PIC) required for SWI/SNF recruitment, and analyze the interactions between PIC components, SWI/SNF and chromatin in an in vitro system. Further, we will explore the contributions of the "histone code" in the remodeling and recruitment of transcription factors to RNR3 using genetic strategies to alter core histone modification states and mutating histone tails. These studies will define mechanisms of transcription factor recruitment, chromatin remodeling and the functions of the TFIID complex that will be applicable to other eukaryotic model systems. [unreadable] [unreadable]